Leukemia is a form of cancer that begins in the blood-forming cells of the bone marrow—soft, inner part of the bones. Leukemia—which literally means “white blood” in Greek—occurs when there is an excess of abnormal white blood cells in the blood. Known as leukocytes, these cells are so plentiful in some individuals that the blood actually has a whitish tinge.
Under normal circumstances, the blood-forming, or hematopoietic, cells of the bone marrow make leukocytes to defend the body against infectious organisms such as viruses and bacteria. But if some leukocytes are damaged and remain in an immature form, they become poor infection fighters that multiply excessively and do not die off as they should. The leukemic cells accumulate and lessen the production of oxygen-carrying red blood cells (eythrocytes), blood-clotting cells (platelets), and normal leukocytes. If untreated, the surplus leukemic cells overwhelm the bone marrow, enter the bloodstream, and eventually invade other parts of the body, such as the lymph nodes, spleen, liver, and central nervous system (i.e. brain, spinal cord). In this way, the behavior of leukemia is different than that of other cancers, which usually begin in solid organs and may spread to the bone marrow.
There are more than a dozen varieties of leukemia, but the following four types are the most common: acute lymphocytic leukemia (ALL); acute myelogenous leukemia (AML); chronic lymphocytic leukemia (CLL); and chronic myelogenous leukemia (CML).
Acute leukemias usually develop suddenly, whereas some chronic varieties may exist for years before they are diagnosed.
Leukemia often is thought to be a childhood disease. In fact, leukemia strikes 10 times as many adults as children. The American Cancer Society predicted that about 30,200 new leukemia cases—27,900 adults and 2,300 children—would be diagnosed in the United States during 1999. Acute myelogenous leukemia (AML) is the most frequently reported form of acute leukemia in adults, and approximately 10,100 new cases were anticipated in 1999.
About 41% of the 30,200 latest cases will have chronic leukemia—an estimated 7,800 chronic lymphocytic leukemia (CLL) cases and 4,500 chronic myelogenous leukemia (CML) cases. In addition, hairy cell leukemia (HCL), a slow-growing lymphocytic cancer, will account for about 604 cases (2% of all leukemias). Sadly, it was estimated that approximately 22,100 American adults and children would die of leukemia in 1999.
Acute myelogenous leukemia (AML) is the most common adult form of leukemia, affecting nearly 5 in every 100,000 men each year.
Chronic leukemia, like many other cancers, is a “disease of old age.” The average age of individuals with chronic lymphocytic leukemia (CLL) is roughly 70 years, and the average age of chronic myelogenous leukemia (CML) patients is 40 to 50 years. By contrast, acute lymphocytic leukemia (ALL) is largely a pediatric disease, usually appearing in children who are under 10 years of age.
In general, leukemia affects more men than women throughout the world, although the male:female ratio is highest in CLL patients in Western countries.
In view of leukemia's prevalence as a disease, the need remains for new ways to treat leukemia. As a result, development of an in vitro culture system of leukemic cells that closely approximates the environment found in vivo becomes important. While in vivo leukemic cells enjoy a selective growth advantage over normal haematopoietic cells in leukemic patients, the opposite occurs in vitro.
Murine models are used to study leukemias and the impact of different therapies on the disease. However, differences in physiology between mice and humans render the murine models as inadequate.
Leukemic cells are so difficult to grow and maintain that failure to form any colonies in vitro is a characteristic by which some myeloid leukemias are classified; normal cells have the selective growth advantage in vitro. This advantage is demonstrated by failure to establish long-term Dexter-type suspension cultures of cells of patients with Philadelphia chromosome (Ph1) positive chronic myeloid leukemia. Over a period of weeks or months the number of cells with the Ph1 karotype decreases until it is undetectable and the number of normal mitoses increases to 100%. The relative inability to survive in vitro under these conditions is now being exploited to purge human marrow of residual leukemic cells in order to return it to the patient some days later in the form of a leukemia-free autologous marrow transplant. The cells transplanted are cultured under the conditions that deviates from the bone marrow environment, because the Dexter culture supports cell growth only in two-dimensions.
Despite the propensity for normal cells to outgrow leukemic cells in vitro or for the leukemic cells to die, several methods for growing leukemic cells in short-term culture have been developed. The method most often used involves leukemic colony-formation in soft agar or methylcellulose. The techniques for growing leukemic colonies were first adopted from systems for normal haematopoietic colony-forming progenitors in semi-solid media and then modified to the particular requirements of leukemic cells. Most studies of leukemic colony-formation deal with acute non-lymphoblastic leukemic (ANLL) cells, the prototype leukemic colony assay. Leukemic cells can be obtained from peripheral blood, bone marrow aspirate or biopsy or rarely from a chloroma or spleen. The procedure for preparing leukemic cells for culture entails removal of the acidic heparin which may harm the cells, removal of the patient's serum which may inhibit growth, removal of erythrocytes which make it difficult to see the leukemic cells, and removal of cells which may inhibit leukemic growth such as granulocytes. Sometimes, cells which may stimulate growth, (e.g., T-lymphocytes and monocytes) or which may themselves form colonies that are difficult to distinguish from leukemic colonies are removed.
The second method for growing leukemic cells is in a suspension culture. Most leukemic cells survive for only a few days in culture media with fetal calf serum.
Finally, a third method to support growth of leukemic cells is the xenografting of human cells into sites such as the anterior chamber of the eye of the nude mouse.
It is now apparent that growth of haematopoietic cells and of leukemic cells in vitro and in vivo is the result of complex interactions between colony-stimulating factors, growth factors, and growth factor receptors. These factors may be humoral factors present in vivo in plasma; they may be autocrine factors generated by the leukemic cells themselves and may be paracrine factors that are dependent on cell-to-cell interactions in the haematopoietic microenvironment.
The establishment of long-term bone marrow cell cultures have been attempted using a pre-established stromal cell support matrix where the stromal matrix provides the support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of cells in culture (Naughton et al., “Hematopoiesis on Suspended Nylon Screen-Stromal Cell Microenvironments,” J. Biomech. Eng., 113:171–177 (1991)). This is based, in part, on the discovery that growth of stromal cells on a nylon screen template will sustain active proliferation of cells in culture for longer periods of time than will monolayer systems. The screen template is a planar, essentially two-dimensional matrix having minimal depth which is defined by the thickness of the strands of nylon mesh.
There has been a lack of a consistent cell culture system for growing leukemia cells. A long-term leukemia cell culture system is needed for characterizing the leukemic progenitors, defining molecules that regulate leukemic proliferation and differentiation, and screening potential chemotherapeutic agents.
The present invention is directed to overcoming the deficiencies in the art.